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Fig. 5. SRC64B tyrosine kinase activity is required for the formation or
stability of the DRL-SRC64B complex. (A) Herbimycin A treatment
leads to significantly decreased amounts of SRC64B co-immunoprecipitating with
DRL. Aliquots of Kc cells co-transfected with DRL-HA and SRC64B-MYC expression
constructs were treated for 24 hours with increasing concentrations of
herbimycin A or equivalent volumes of the DMSO carrier, WCEs prepared,
DRL-SRC64B complexes immunoprecipitated with anti-DRL and then immunoblotted
with anti-MYC (SRC64B) antibody (top panels). Lanes 1 and 5, untreated
samples; lanes 2-4, herbimycin A-treated at 2.5, 5 and 10 µM final
concentration, respectively; lanes 6-8, DMSO controls. Equal amounts of WCEs
were evaluated for SRC64B-MYC expression (middle panels) and overall tyrosine
phosphorylation levels (lower panels). (B) Wild-type (WT), but not
kinase-dead (KD), SRC64B interacts with the intracellular domain of DRL
(DRL-intra) in a mammalian two-hybrid assay. The indicated fusion protein
constructs were transfected into SFK-deficient cells and luciferase activity
was measured 48 hours post-transfection and plotted, normalized to an internal
control. Immunoblotting for DRL and SRC64B species (inset) indicates
equivalent expression of the test plasmids. An irrelevant lane between lanes 5
and 6 was removed in preparing the panel.
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